Although famous for secreting the cerebrospinal fluid (CSF), the choroid plexus has drawn relatively little attention from basic and clinical scientists. Every day, human choroid plexus epithelial cells (CPECs) secrete about two cups of protein-rich CSF, which clears out waste products from and delivers beneficial molecules to every cell in the brain and spinal cord. CPEC defects have been implicated in a large number of major CNS disorders, including hydrocephalus, psychiatric conditions, and neurodegenerative diseases. However, almost all of the literature related to CPEC biology and disease is descriptive due to a lack of CPEC-targeted tools. Importantly, if such tools existed, delivery of compounds to CPECs - in either experimental animals or patients - is straightforward, because CPECs exchange freely with the peripheral circulation. This greatly simplifies drug delivery and strengthens the appeal of CPECs as a new target for CNS studies and disease therapies. Despite this appeal, CPEC-targeted drug screens have not been possible due to difficulties in propagating and deriving them in culture. However, we recently developed a method for generating derived CPECs (dCPECs) from mouse and human embryonic stem cells. This method provides, for the first time, a scalable means to produce dCPECs in the large numbers required for high-throughput screening (HTS). In this R21 proposal, we combine the dCPEC technology inventor with experts in assay development and drug screening to develop a high-throughput dCPEC screening platform. In preliminary studies, we describe dCPEC generation, candidate cell lines to be screened for HTS compatibility, and a sensitive 96-well ELISA for secreted human TTR, an ideal proxy for CPEC secretion and an important molecular target in its own right. The TTR ELISA will be converted into an HTS-compatible AlphaLISA, which will be used for a pilot screen to determine assay readiness. Hits and analogs from the pilot screen will then be screened and validated via testing funnels that include new and already-established assays of CPEC function, mechanism, and specificity. This validated high-throughput dCPEC platform will set the stage for full-scale screening proposals to generate first-in-class tool and therapy leads for a brand-new CPEC-based biomedicine.